Improvements to in-line desalting of oligosaccharides separated by high-pH anion exchange chromatography with pulsed amperometric detection.
High-pH anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD) (1) is routinely used to separate neutral and charged oligosaccharides differing by branch, linkage, and positional isomerism. Oligosaccharides are eluted in 0.1 M NaOH with gradients of sodium acetate (up to 0.25 M). Analyses of HPAEC/PAD-purified oligosaccharides generally require neutralization and removal of eluent salts. To facilitate the process, we designed and produced a cation-exchange system to remove sodium ions (Na+) from the eluent after oligosaccharide detection [the Carbohydrate Membrane Desalter (CMD), with a volatile regenerant]. Exchange of >99.5% of eluent Na+ for hydronium ions (H3O+) within the CMD generates dilute acetic acid (removable by vacuum evaporation). The exchange process desalts up to 0.35 M Na+ at 1.0 ml/min. Oligosaccharides collected after on-line desalting, evaporated and resuspended in their original volume of deionized water contained < or = 350 muM residual Na+ when the eluting sodium concentration was 300 mM. This represents a desalting efficiency of >99.8%. Recovery of neutral and sialylated oligosaccharides under these conditions ranged from 75 to 100%. With the CMD system and postcollection evaporation, HPAEC/PAD can purify oligosaccharides ready for further characterization. As a proof test, oligosaccharides from a human monoclonal antibody were separated by HPAEC/PAD, desalted with the CMD system, dried, and analyzed by matrix-assisted laser desorption-ionization, time-of-flight mass spectrometry.
Thayer
J R
JR
Dionex Corporation, Sunnyvale, California.
Rohrer
J S
JS
Avdalovic
N
N
Gearing
R P
RP
D000085:Acetates / Q000378:metabolism; D002236:Carbohydrate Conformation; D002852:Chromatography, Ion Exchange / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008566:Membranes / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012492:Salts; D012964:Sodium / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013431:Sulfates / Q000378:metabolism
D000085:Acetates; D009844:Oligosaccharides; D012492:Salts; D013431:Sulfates; D012964:Sodium